Lectures 10-14: Endosymbiosis and Organelles

Question 1

What is primary, secondary and tertiary endosymbiosis?

Answer 1

Primary: I prokaryote, 1 membrane

Secondary: A organism consumes another with an endosymbiont (2 membranes)

Tertiary: 3+ membranes

Question 2

True or false, a chloroplast has only a few copies of cpDNA?

Answer 2

False, each organelle has many copies. There is about the same amount of DNA present in organelles (combined) as the nucleus

Question 3

Give two methods for studying organelles and their genomes with comparative genomics

Answer 3

• compare organellar genomes to those of bacteria. Can learn about impact of endosymbiosis on the eukaryotic nuclear genome.
• Compare organellar genomes to nuclear genomes of different species. Can tell us how organellar DNA evolves and migrates to the nucleus over shorter timescales.

Question 4

How do you study endosymbiotic gene transfer in the lab?

Give the example with the tobacco plant

Answer 4

Experimental systems involving transgenic organisms can be used to observe EGT in real time. Can provide insight into the mechanisms and frequency of EGT. Experiment in tobacco plant

1. Link a gene to a “plastid-specific” promoter to confer resistance to spectinomycin
2. Link another gene to “nucleus specific” promoter to confer resistance to kanamycin (still in plastid)
3. Resistant to only plastid specific drug
4. Screen for kanamycin resistance, if it grows it means that the gene has migrated to the nucleus and the nucleus-specific promoter containing gene can be expressed.

Question 5

True or false. Endosymbiotic gene transfer takes place over different timescales

Answer 5

The mass migration of DNA from organelle to nuclear runs at a very variable rate (true!)

• Ancient transfers (eg. during evolution from mitochondrial/plastid progenitors)
• Somewhat recent transfers (organelles have different size genomes in different species)
• Very recent transfer (usually giving rise to non-functional DNA fragments continuously - eg. in tobacco)

Question 6

How widespread is endosymbiotic gene transfer?

Answer 6

Very!

The mitochondrial genome has become very small in most organisms (sometimes to nothing), the plastid genome is even smaller (sometimes to nothing as well).

Both proteomes are usually under 1000 proteins! The proteins that both organelles need are provided by the nuclear genome. If the organelle provides any genes, it is quasi-autonomous.

Question 7

Describe the genomic ‘footprint’ of endosymbiosis in Arabidopsis thaliana

Answer 7

• Almost 25% of the nuclear genome is from the cyanobacterial progenitor of the plastid. So there is a huge footprint because of extensive EGT.
• Nucleus encoded cyanobacterium derived proteins are targeted to different subcellular locations (not just the plastid)

Question 8

Define EGT

Answer 8

Endosymbiotic gene transfer - The process by which fragments of endosymbiont/organelle DNA ends up in the nuclear genome. Doesn’t need to be intact genes!

Question 9

Define NORGs

Answer 9

Nuclear integrant of organellar DNA Includes:

• NUMTs
• NUPTs

We can say very confidently that these fragments come from their respective organelles.

Question 10

Define NUMTs

Answer 10

Nuclear mitochondrial DNA

• Mitochondrial DNA fragments integrated into the nuclear genome

Question 11

Define NUPTs

Answer 11

Nuclear plastid DNA

• Plastid DNA fragments integrated into the nuclear genome.

Question 12

Define NUNMs

Answer 12

Nuclear nucleomorph DNA
- Fragments of DNA from nucleomorph genomes integrated into the nuclear genome (limited to single celled algae with nucleomorphs obviously)

Question 13

True or false? The total amount and density of NUMTs and NUPTs varies a lot!

Answer 13

True!

The total amount varies between mitochondria and plastids in the same organism!

Eg. Arabidopsis has lots of NUMTs but not nearly so many NUPTsThe amount can even vary among organelles in an organism.

Question 14

Describe how the Domestic Cat provides an example of NUMTs

Answer 14

• An 8 kb NUMT migrated to the nuclear genome and hybridized with a nuclear chromosome and duplicated massively there as a tandem repeat
• The spread is mediated by recombination

Question 15

In humans, did NUMTs arise before or after the divergence of humans and chimpanzees? Why is it hard to answer this question?

Answer 15

Before AND after

It is not clear how many human NUMTs represent unique NUMT transposition/transfer events vs. duplications of previous transposition events.

Because of this, there is lots of NUMT variation even among individuals! It is hard to count NUMTs because they can jump into the genome and then jump around them.

NUMTs also disappear fairly quickly, they acquire mutations and deletions at a rate similar to pseudogenes. So they are only detectable for a short amount of time.

When there is a lot of EGT, it is hard to determine if you are just reading background noise (from lysed organelles) or true nuclear genes.

Question 16

True or false, NUMTs can cause disease. Why or why not?

Answer 16

True

eg. putting a premature stop codon into an important nuclear gene will result in a nonfunctional protein. This can be caused by DNA damage (eg from radiation), making the genome more accepting of foreign elements/recombination. Spontaneously. Or the diseases can be inherited if the insertion occurs in the germ line.

Question 17

True or false, NUMTs can go on to become active elements of the nuclear genome?

Answer 17

True.

In Bigelowiella natans, a NUMT inserted into a gene and became an active intron. Because it is spliced out, no harm done!

Question 18

Is there a correlation between number of organelles in a cell and endosymbiotic gene transfer prevalence?

Answer 18

Yes.

More organelles = more transfer events

Organelle lysis is the main mechanism for DNA transfer from organelle to nucleus.

Question 19

How much DNA is usually transferred in endosymbiotic gene transfer?

Answer 19

Any fragments can be transferred in principle. From ten bp fragments to an entire organellar genome.

Question 20

How is organellar DNA integrated with nuclear DNA? Is this random and can only DNA be integrated?

Answer 20

NUMTs and NUPTs are incorporated non-homologously.

Non-homologous end joining at sites of double-strand break (DSB) repair. Integration appear to be non-random (eg. into A/T rich regions more)

cDNA from organellar RNA can also be integrated Summary: DNA damage followed by DSB repair with organellar DNA

Question 21

What is the fate of transferred DNA (and the donor DNA in the organelle) after EGT?

Answer 21

• Majority of EGTs are non-functional: they quickly become pseudogenes and disappear due to mutations
• Donor DNA continues to exist in the organelle OR if the gene is successful in the nucleus, it will be deleted from the organellar genome.

Question 22

Giv e three qualities of a good potentiation insertion site for EGT

Answer 22

• A+T rich
• Highly bendable
• Open chromatin

Question 23

How are the products of genes transferred from the organelle to the nucleus imported to the organelle?

Answer 23

• N-terminal signal sequences (transit peptides) bind with receptors on surface of organelle
  
  • Protein machinery (TOMs/TIMs/TOCs/TICs) mediate translocation of the protein - Transit peptide removed once protein is inside the organelle

Question 24

Name the translocons of mitochondria and chloroplasts?

Answer 24

Mitochondria: TOMs and TIMs

Chloroplasts: TOCs and TICs

Question 25

What three elements are needed for a NUPT or NUMT to provide proteins for their organelle?

Answer 25

• Promoter
• Transit peptide
• Terminator

Question 26

When does an endosymbiont become an organelle?

Answer 26

When both endosymbiotic gene transfer AND protein import can take place (ie. a dedicated protein import apparatus is in place.

Question 27

Is the nucleus a membrane bound organelle?

Answer 27

No, because the nuclear envelope is continuous with the endoplasmic reticulum.

Question 28

Describe the case of Paulinella chromatophora and how its chromatophores relate to plastids

Answer 28

It is a single celled eukaryote with two sausage like chromatophores, which divide synchronously with the host cell. The host and chromatophores cannot grow apart from one another.

• Chromatophores evolved from cyanobacteria independent of plastids
• The chromatophore genome appears to be undergoing reduction (lots of pseudogenes, which are uncommon in prokaryotes)
• Plastids are about a billion years old, this endosymbiosis is only about 60 million years old

Question 29

Is the chromatophore of paulinella chromatophora an endosymbiont or an organelle?

Answer 29

Organelle
- There is EGT occuring, with genes of chromatophore origin in the host nuclear genome

• Targeting of nucleus encoded proteins to the chromatophore has been observed

So EGT + protein import satisfies organelle requirements

Question 30

Is Buchnera an endosymbiont of aphids? Or organelles?

Answer 30

EGT has taken place but there is no FUNCTIONAL gene transfer or protein import taking place: endosymbiont Though, there is one gene (RlpA4) which encodes a lytic transglycosylase with an N-terminal signal peptide to target to the ‘endosymbiont/organelle’Blurry distinction between endosymbiont and organelle in this case. Perhaps organelle in the making.

Question 31

How do transferred genes acquire gene expression and targeting elements?Give four methods

Answer 31

They acquire them de novo, but they can also acquire them from pre-existing genes:

• De novo acquisition
• Insertion into duplicated copies of other genes (steals elements from a non-necessary gene copy)
• Insertion into a tandem duplication (one of the two is dispensable)
• Insertion into an intron, with co expression by alternative splicing.

Question 32

Describe the key features of the gene-transfer ratchet

Answer 32

• EGT is unidirectional
  
  • EGT is accidental and inevitable
  • Intact genes on fragments will sometimes (rarely) be expressed and even more rarely inserted downstream of a region encoding a N-terminal targeting sequence, allowing protein import.
  • If the nuclear copy of the gene is lost, evolution can try again
• If gene B is lost from organellar genome, it is lost forever from that genome and the nuclear copy becomes essential for survival. After time, the nuclear genome will ultimately incorporate all organellar genes that can potentially function there

Question 33

Give three hypothesis for the retention of organellar genomes

Answer 33

• Hydrophobicity hypothesis: organellar genome coded proteins are hydrophobic, making them hard to import across the membranes of the organelle (very true for mitochondria, less true for plastids)
• Redox regulation hypothesis: Electron transport proteins must be quickly directedly regulated by redox state of mitochondria, in cases where the mitochondrial genome has disappeared, the electron transport chain is no longer used.
• Genetic code disparity hypothesis: There is always at least one disparity between a cell and the organellar genome’s genetic code (doesn’t hold up for all cases)

Question 34

What are the two main types of plastids?

Answer 34

Primary plastids: evolved directly from cyanobacteria and surrounded by two membranes.

Secondary plastids: Acquired indirectly from primary plastid-bearing algae, surrounded by three or four membranes.

Secondary plastids have evolved at least 3 times (and probably more): both red and green alga endosymbionts have been involved).

Question 35

What does plastid protein import say about the evolution of plastids?

Answer 35

Some TOC and TIC proteins in algae and plants are still encoded by the plastid genome and many have homologs in cyanobacteria.

This conservation is a clear hint towards common ancestry between plastids and cyanobacteria.

Question 36

List some conserved structural features of plastid genomes

Answer 36

• Circular (presumed)
• Genes on both strands (bacterial type operons clearly present)
• Inverted repeats (incl. ribosomal operon (yellow)), sometimes including large stretches of non-coding DNA
• Large and small regions with single-copy genes

All these are indicative of an early prokaryotic ancestor.

Question 37

Describe the structural diversity of plastid genomes

Answer 37

• Inverted repeated (coding for rRNA operon) is highly conserved
• The genomes of non-photosynthetic plastids are much reduced in size but generally conserved in structure

Question 38

Dinoflagellates have very small plastid genomes, why?

Answer 38

• Has a tiny circular chromosome with 0-5 genes and repetitive ‘core’ sequences involved in minicircle replication (and recombination between circles)

Where are the missing genes?: At least some of the genes are in the nuclear genome, but we don’t know yet for sure.

Question 39

What type of plastids have the largest genomes?

Answer 39

Red algal and red algal-derived secondary plastids

Question 40

What are plastid genomes mostly composed?

Answer 40

• Photosynthetic proteins
• Translation proteins (but not transcription)

That’s about it, all known plastid genomes are significantly reduced compared to cyanobacterial genomes.

Question 41

Describe how the nuclear genome works in tandem with the plastid genome on photosynthesis.

Answer 41

• Components of the light harvesting apparatus, electron transport chain and ATP synthase complex are a mixture of nucleus and plastid encoded genes
• It is therefore not the case that some biochemical processes in the plastid are carried out exclusively by proteins encoded on the plastid genome (this applies to all type of plastids)

Question 42

Describe the ultrastructure and protein import capabilities of secondary plastids

Nucleomorph?

Answer 42

• The extra membrane surrounding secondary plastids complicates the process of protein import
• Such organisms use the signal peptide secretion system to begin the process of targeting nucleus-encoded proteins across the multuple membranes - they have bipartile N-terminal leader sequences (containing SPs and TPs)
• Some complex algae have a nucleomorph (which has genes) between the inner and outer pairs of plastid membranes (periplastidial compartment / PPC), which is the former cytosol of the algal endosymbiont. The nucleomorph provides some proteins for the PPC
• Such cells have four DNA containing compartments and two cytosolic compartments

Question 43

Describe nucleomorph genomes

Answer 43

• Tiny
• Dense
• Weird (small GC content, ultra fast evolving with lots of ORFans)
• Reductive evolution (seen in bacteria/plastids/mitochondria), despite the eukaryotic origin of the nucleomorph

Question 44

What do nucleomorph genomes encode?

Answer 44

• House keeping proteins (eg. PPC and plastid are heavily reliant on import of nucleus encoded proteins, PPC ribosomes are built from nucleomorph and host nucleus encoded proteins)
• Plastid proteins (though incomplete set)
• NO cytosol enzymes for core metabolic functions (all moved to nuclear genome)

Question 45

Why do nucleomorphs persist?

Answer 45

They have actually vanished from most other complex algae

But they persist because:
- It appears like plastid/nucleomorph genes migrate to the nucleus less readily than mitochondrial genes

NUNMs: nuclear nucleomorph genes

Question 46

What is the limited transfer window hypothesis?

Answer 46

There is a correlation between the number of organelles per cell and the frequency of EGT from that organelle.

Therefore, we may say that there could be a transfer window that remains open for a set period of time before shutting. Those with more organelles have more chances of transfer.

In organisms where the nucleomorph has disappeared, it could be because the transfer window was open long enough for all nucleomorph DNA to migrate.

more evidence: polyplastididc species have 80 times more NUPTs than monoplastidic species and never have nucleomorphs (seen yet)

The nucleomorph genomes of cryptomonads and chlorachniophytes (monoplastidic species) appear ‘frozen’ at the moment.